Over the past 10 years, our laboratory has made major advances in understanding how nosocomial pathogens that colonize the gut following surgical injury cause lethal sepsis. We discovered that Pseudomonas aeruginosa, a human opportunistic pathogen that colonizes the gut of as many as 50% of critically ill patients, shift its phenotype from that of indolent colonizer to lethal pathogen in direct response to host stress compounds released into the gut during surgical injury that include: 1.) immune elements (interferon 3), 2.) opioids (morphine, dynorphin), and 3.) end-products of cellular hypoxia (adenosine). Most recently we demonstrated that phosphate depletion also develops at intestinal sites of P. aeruginosa colonization during surgical injury and enhances the responsiveness of P. aeruginosa to host stress compounds via highly sensitive phosphate regulatory mechanisms that intersect with the quorum sensing system, the major mechanism of virulence gene activation in P. aeruginosa and other bacteria. We also discovered that in a phosphate rich environment, P. aeruginosa virulence expression is markedly attenuated in the presence of host stress compounds and its lethal effect in the mouse intestine completely abrogated during surgical injury. We now propose to accomplish 4 aims to extend our ongoing work to its natural translational and therapeutic endpoint that include: 1.) determining the molecular mechanisms by which phosphate depletion and host-tissue BSCs synergize to shift P. aeruginosa into a lethal trajectory in the intestine following surgical injury 2. ) determining the site-specific molecular mechanisms involved in P. aeruginosa virulence activation in the intestinal tract in vivo following surgical injury. 3.) designing phosphate rich synthetic mucins that molecularly silence P. aeruginosa when present in the intestinal tract during surgical injury. 4.) determining the mechanisms by which intestinal contents from critically ill humans shift the molecular signature of P. aeruginosa into a lethal trajectory using custom PCR arrays. Results from this proposal will provide an unprecedented level of molecular detail on the pathogenesis of lethal gut-derived sepsis and provide novel therapeutic direction for the discovery and testing of compounds that interfere with virulence activation of lethal pathogens that colonize the gut of critically ill patients. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the molecular basis by which Pseudomonas aeruginosa, a common pathogen colonizing the gut of surgically injured patients, shifts its phenotype from indolent colonizer to lethal pathogen as a result of local environmental cues present in the intestine during surgical injury. Using nematodes, mice, and human intestinal contents from critically injured patients, we will elucidate the molecular basis by which P. aeruginosa expresses a lethal phenotype against the intestinal epithelium and identify novel therapeutic targets and non-antibiotic compounds that molecularly silence this pathogen from expressing a lethal phenotype.